A high-temperature water vapor equilibration method to determine non-exchangeable hydrogen isotope ratios of sugar, starch and cellulose

Abstract

The analysis of the non-exchangeable hydrogen isotope ratio (δ2Hne) in carbohydrates is mostly limited to the structural component cellulose, while simple high-throughput methods for δ2Hne values of non-structural carbohydrates (NSC) such as sugar and starch do not yet exist. Here, we tested if the hot vapor equilibration method originally developed for cellulose is applicable for NSC, verified by comparison with the traditional nitration method. We set up a detailed analytical protocol and applied the method to plant extracts of leaves from species with different photosynthetic pathways (i.e., C3, C4 and CAM). δ2Hne of commercial sugars and starch from different classes and sources, ranging from −157.8 to +6.4‰, were reproducibly analysed with precision between 0.2‰ and 7.7‰. Mean δ2Hne values of sugar are lowest in C3 (−92.0‰), intermediate in C4 (−32.5‰) and highest in CAM plants (6.0‰), with NSC being 2H-depleted compared to cellulose and sugar being generally more 2H-enriched than starch. Our results suggest that our method can be used in future studies to disentangle 2H-fractionation processes, for improving mechanistic δ2Hne models for leaf and tree-ring cellulose and for further development of δ2Hne in plant carbohydrates as a potential proxy for climate, hydrology, plant metabolism and physiology.